Genomic Characterization of Multidrug-Resistant Extended Spectrum β-Lactamase-Producing Klebsiella pneumoniae from Clinical Samples of a Tertiary Hospital in South Kivu Province, Eastern Democratic Republic of Congo

Multidrug-resistant (MDR) and extended spectrum β-lactamase (ESBL)-producing extra-intestinal K. pneumoniae are associated with increased morbidity and mortality. This study aimed to characterize the resistance and virulence profiles of extra-intestinal MDR ESBL-producing K. pneumoniae associated with infections at a tertiary hospital in South-Kivu province, DRC. Whole-genome sequencing (WGS) was carried out on 37 K. pneumoniae isolates displaying MDR and ESBL-producing phenotype. The assembled genomes were analysed for phylogeny, virulence factors and antimicrobial resistance genes (ARG) determinants. These isolates were compared to sub-Saharan counterparts. K. pneumoniae isolates displayed a high genetic variability with up to 16 sequence types (ST). AMR was widespread against β-lactamases (including third and fourth-generation cephalosporins, but not carbapenems), aminoglycosides, ciprofloxacin, tetracycline, erythromycin, nitrofurantoin, and cotrimoxazole. The blaCTX-M-15 gene was the most common β-lactamase gene among K. pneumoniae isolates. No carbapenemase gene was found. ARG for aminoglycosides, quinolones, phenicols, tetracyclines, sulfonamides, nitrofurantoin were widely distributed among the isolates. Nine isolates had the colistin-resistant R256G substitution in the pmrB efflux pump gene without displaying reduced susceptibility to colistin. Despite carrying virulence genes, none had hypervirulence genes. Our results highlight the genetic diversity of MDR ESBL-producing K. pneumoniae isolates and underscore the importance of monitoring simultaneously the evolution of phenotypic and genotypic AMR in Bukavu and DRC, while calling for caution in administering colistin and carbapenem to patients.


Introduction
Infections caused by MDR Gram-negative bacteria (GNB) are becoming a major public health issue all over the world. Because of the limited antimicrobial treatment options, these infections are frequently associated with high morbidity, prolonged hospitalization, high healthcare costs, and poor outcome [1]. These bacteria include MDR ESBL-producing Enterobacteriaceae, which have been identified as a major contributor to the current AMR crisis by the World Health Organization (WHO) [2]. According to data from Asia, Latin America, Europe, and Africa, the dramatic increase in third-generation cephalosporin resistance in Enterobacteriaceae is mainly due to the spread of bla CTX-M-15 gene ESBL [3][4][5], with bla CTX-M-15 gene being the most common ESBL gene worldwide [6,7]. While most studies of MDR ESBL-producing Enterobacteriaceae have focused on Escherichia coli [8][9][10][11][12][13][14][15], few studies have characterized the whole genome of MDR ESBL-producing Klebsiella pneumoniae in Africa, even though this agent is one of the most widespread MDR ESBL-producing Enterobacteriaceae associated with extra-intestinal infections both in hospital and community settings [16][17][18][19][20][21]. Moreover, several strains of K. pneumoniae have been linked to multinational epidemics all over the world [21,22]. In the DRC, there is little epidemiological data on K. pneumoniae and its role in the emergence of hospital and community-acquired infections. Nonetheless, the few available studies that have been conducted in the country show an increasing prevalence of MDR K. pneumoniae in hospital-acquired infections [16,17,23]. We previously reported a high prevalence of MDR ESBL-producing E. coli, K. pneumoniae, and Enterobacter cloacae in the urinary tract and bloodstream infections in eastern DRC [16,17]. WGS performed later on MDR ESBL-producing ExPEC isolates from the same collection showed the high prevalence of the ST131 and ST405 pandemic clones carrying the plasmid-mediated CTX-M-15 gene as the primary cause of ESBL-production in EXPEC [9]. Accordingly, the purpose of this study was to characterize extra-intestinal EBSL-producing K. pneumoniae isolates from the South-Kivu province of DRC using WGS. These isolates were collected in the years 2013 through 2017. The WGS data allowed phylogenomic analyses, as well as the characterization of the virulome and the resistome.

Ethical Clearance
The study protocols were approved by the Ethical Review Board (ERB) of the Université Catholique de Bukavu, DRC (UCB/CIES/NC/001/2023). The ERB granted a waiver of informed consent after deciding that the study complied with international guidelines on antibiotic surveillance studies. Samples were analysed anonymously to protect confidentiality.

Bacterial Strains
Between September 2013 and June 2017, GNB (n = 2051) were isolated from patients who presented to the 385-bed General Hospital of Bukavu (South Kivu province, DRC) with urinary tract infections (n = 1608), bloodstream infections (n = 416), as well as peritonitis (n = 12), pleurisy (n = 7), and soft tissue infections (n = 8). GNB bacteria were isolated and identified at the species level in urine samples as previously described [16]. Likewise, GNB associated with bloodstream, peritoneal, pleural, and soft skin tissue infections were isolated and identified at the species level following the protocol established in 2014 at HGPRB [17]. All the K. pneumoniae isolates (n = 449) from this 2013-2017 collection represented 21.9% of all extra-intestinal GNB isolates. They were selected for additional analyses, including antimicrobial susceptibility testing.

Antimicrobial Susceptibility Testing
K. pneumoniae isolates were tested for susceptibility to 14 antimicrobial agents (i.e., amikacin, gentamicin, ampicillin, amoxicillin, amoxicillin/clavulanic acid, cefepime, ceftriaxone, ceftazidime, cefotaxime, ciprofloxacin, imipenem, meropenem, nitrofurantoin, and colistin) by the method of minimum inhibitory concentration (MIC). The MIC for each of the 14 antimicrobial agents minus colistin was determined after 16-20 h incubation on Mueller-Hinton plates inoculated with suspensions of isolates at a fixed density (0.5 to 0.6 McFarland standard), using MIC test strips (Liofilchem, Roseto degli Abruzzi, Italy) according to the manufacturer's recommendations. MIC for colistin was determined by the broth microdilution method, using the ComASP ® Colistin 0.25-16 µg/mL panel (Liofilchem, Roseto degli Abruzzi, Italy) according to the manufacturer's recommendations. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, which were updated in January 2022, were used to interpret the susceptibility or resistance status of each isolate. ESBL-production assay was performed by using the MIC Test Strip ESBL Ceftazidime (CAZ)/Ceftazidime + Clavulanic acid (CAL) panel (Liofilchem, Roseto degli Abruzzi, Italy) according to the manufacturer's recommendations). E. coli ATCC 35218 and K. pneumoniae ATCC 700603 strains were used as ESBL-negative and positive controls, respectively, while E. coli ATCC 25922 and E. coli NCTC 13846 were used as colistin-susceptible and resistant strains, respectively.

Bacterial DNA Extraction
K. pneumoniae isolates were cultured overnight in 10 mL Luria-Bertani broth. Genomic DNA was isolated using the EZ1 Advanced XL Biorobot and the tissue DNA kit (Qiagen, Hilden, Germany) with the Bacterial card, according to the manufacturer's instructions. For each isolate, genomic DNA was quantified using Qubit ® fluorometric quantitation (ThermoFisher Scientific, Hillsboro, OR, USA) and normalized to 0.2 ng/µL.

Library Preparation and Next Generation Sequencing
The library was prepared using the Nextera XT DNA Library preparation kit (Illumina, San Diego, CA, USA) as previously described [12]. Briefly, bacterial DNA was fragmented, and then tagged with sequencing adapters using the Nextera transposome (Nextera XT DNA Library Preparation Kit, Illumina, San Diego, CA, USA). PCR reaction of 12 cycles was performed to amplify the DNA fragments and to incorporate indices for dual-indexed sequencing of pooled libraries. The 1 nM pooled library was denaturated and diluted prior to loading on a MiSeq paired-end 2 × 150 (MiSeq reagent kit V2 (300 cycles) or 2 × 300 base pairs (bp) (MiSeq reagent kit V3 (600 cycles) sequence run.

Genome Assembly, Annotation, and Alignment
Raw sequencing data from all K. pneumoniae isolates were submitted to the European Nucleotide Archive (ENA, http://www.ebi.ac.uk/ena; submitted on 22 December 2022) and are available under accession number PRJEB56212. These data were analysed using the following bioinformatics pipeline. All reads were quality checked using FastQC v.0.11.9 [24], and processed using trimmomatic v.0.39 [25]. Paired-end reads from each K. pneumoniae isolate were assembled de novo using the Spades v.3.13.0 algorithm [26] to generate a draft genome sequence for each isolate. Quality assessment of the genome assemblies was performed using QUAST 5.0.2 [27]. In addition, the quality and completeness of the genome assemblies were confirmed using the BUSCO v.4.1.1 algorithm and the bacteria_odb10 dataset [28] The PATRIC database [29] was used to identify complete or draft genomes of other K. pneumoniae strains isolated from patients in other sub-Saharan African countries. In-silico MLST typing was performed on all genomes by using the screen-Blast-mlst function of the Pathogenomics home-made R package (https://github.com/UCL-CTMA/Pathogenomics) and the K. pneumoniae allele sequences and MLST profiles available in the BIGSdb-Pasteur platform (https://bigsdb.pasteur.fr/klebsiella; accessed on 22 April 2022). It should be noted that the assignment of an allele type requires a perfect match with the corresponding sequence (i.e., 100% sequence identity and coverage).
All genomes were submitted to kSNP3.0 [30] using the default settings, with the exception of a k-mer size of 19 and the '-core' option. This software identified SNPs without the need for genome alignment or a reference genome. The phylogenomic tree based on the core genome was visualized using the R package ggtree [31]. The Pathogenomics custom R package was also used to screen all genomes for the virulence factor genes described in the VFDB database (http://www.mgc.ac.cn/VFs/; accessed on 24 August 2022 ) [32] and which are specifically associated to K. pneumoniae. The thresholds for sequence identity and coverage were respectively set at 80% and 50%. The R package ggtree was used to display the coverage percentage of each virulence gene in grayscale heatmaps. In silico K-typing and O-typing were performed on draft genomes of the 37 K. pneumoniae isolates, using Kleborate (v.2.2.0) [33] and Kaptive (v.2.0.3) [34]. Each isolate was assigned a virulence score based on the presence or absence of the yersiniabactin (ybt), colibactin (clb), and aerobactin (iuc) genes, as previously described [33].
Simultaneously, each draft genome was screened for the presence of AMR genes and point mutations using the AMRFinderPlus 3.10.5 [35] tool developed by the National Center for Biotechnology Information (NCBI). The draft genomes were screened for the presence of plasmid incompatibility groups, in particular the IncF group. The Replicon Scheme Typing (RST) developed by Villa et al. [36] was used for classification of IncF plasmids according to the FAB (FII, FIA, FIB) formula. The DNA sequence of the novel FIA replicon was submitted to the curator of the pMLST database (https://pubmlst.org/plasmid/; accessed on 26 July 2022) for ST assignment.

Bacterial Strains
A set of MDR ESBL-producing K. pneumoniae isolates were identified from the total K. pneumoniae (n = 89/449,~20%) isolated from the GNB collection assembled between 2013 and 2017 at the Bukavu General Hospital, South Kivu, DRC. Due to isolate loss, either because of a problem with isolate storage or other technical issues, only 37 of the original 89 MDR ESBL-producing K. pneumoniae isolates were thoroughly analysed. The isolates were not duplicate clinical samples, but rather one sample per patient. The majority (n = 31, 83.8%) of the isolates were cultured from samples obtained from patients who were hospitalized and included all isolates from bloodstream (n = 19, 51.4%), urinary tract (n = 9, 24.3%), peritoneal fluid (n = 2, 5.4%), and soft tissue (n = 1, 2.7%). The remaining (16.2%) isolates (n = 6) were collected from patients with community-acquired urinary tract infections.

Antimicrobial Susceptibility Testing
All K. pneumoniae isolates (n = 37) were susceptible to imipenem, meropenem, and colistin while showing reduced susceptibility to ampicillin, amoxicillin, cefotaxime, ceftazidime, and ceftriaxone. While the majority of them (30 out of 37) showed reduced susceptibility to cefepime, they all exhibited the ESBL-production phenotype (Tables 1 and 2).

Phylogenomic Analysis
SNP-based phylogenetic analysis of the MDR ESBL-producing K. pneumoniae from DRC revealed a high level of genetic diversity. The 37 K. pneumoniae isolates were distributed in 16 different clusters (Figure 1), each of which corresponded to a unique MLST profile ( Table 3).

Detection of AMR Point Mutations
In addition to AMR genes, AMR point mutations were identified (Figure 3). ST340 isolates (CTMA_1653, CTMA_1674, CTMA_1676, and 1678), and the ST11 isolate (CTMA_1688) carried the substitution S83I in the quinolone resistance-determining region (QRDR) of the gyrase A gene (gyrA), as well as the S80I substitution in the QRDR of the Deoxyribonucleic acid topoisomerase IV subunit A gene (parC). The two ST15 isolates CTMA_1657 and CTMA_1668 both had the S83F and D87A substitutions in gyrA, as well as the S80I substitution in the parC.
In ST2094 (CTMA_1682) and ST16 (CTMA_1689) isolates, the gyrA gene harboured the S83F and D87N substitutions, while parC had the E84K or S80I substitution. Likewise, ST874 isolates CTMA_1663 and CTMA_1667 both harboured the S83Y and D87A substi-tutions in gyrA as well as the S80I substitution in parC.
In the gyrA gene of five ST48 isolates, including CTMA_1650, CTMA_1661, CTMA_1662, CTMA_1666, and CTMA_1672, the S83Y substitution was the only mutation. The QRDR of parC from these isolates lacked any additional mutations.

Discussion
Being the cause of frequent outbreaks in hospitals, K. pneumoniae is becoming a significant global cause of severe infections. Despite reports of a high prevalence of MDR K. pneumoniae in a tertiary hospital in the eastern city of Bukavu in the DRC [16,17,22], neither this province nor the entire DRC have yet carried out molecular characterization of MDR ESBL-producing K. pneumoniae. We believe that this WGS study is the first to give insights on the genomic characteristics of MDR ESBL-producing K. pneumoniae isolates in this district of South-Kivu, eastern DRC. According to the WGS results, the MDR ESBL-producing K. pneumoniae isolates from clinical samples collected at the Bukavu hospital between 2013 and 2017 were found to have comparable ST to those commonly reported elsewhere.
The absence of carbapenemase genes, however, makes the current isolates significantly different from the global lineages, whose susceptibility to carbapenems is known to be decreased by these genetic determinants. A widespread use of carbapenems to treat severe ESBL-associated infection is likely too new in Bukavu to have seen the emergence of pandemic MDR ESBL-producing carbapenem-resistant K. pneumoniae (CRKP) lineages. This hypothesis is consistent with other data on MDR ESBL-producing K. pneumoniae in Ghana [46] and in southwestern Nigeria [47], where the administration of carbapenems was still restricted at the time of collection of isolates. In any case, the current results highlight the potential risk of a routine nontargeted administration of antimicrobial carbapenems, which is becoming more common in the province as a factor favouring the rapid emergence of carbapenem-resistant K. pneumoniae lineages.
When compared to several genomes from other sub-Saharan African countries, the higher content of virulence genes in the current eastern DRC K. pneumoniae isolates is a third difference (Figure 1). It is worth noting, however, that no hypervirulence gene was found in the eastern DRC isolates. This result is consistent with other data describing nonhypervirulent MDR ESBL-producing K. pneumoniae in hospital-acquired infections.
An intriguing finding is the presence of mutations associated with resistance to colistin in otherwise colistin-susceptible MDR ESBL-producing isolates. This observation was indeed made on isolates collected from patients whose treatment did not include colistin and in a province where there is no record of colistin administration. Regarding the use of colistin, these results should raise concerns about a potential rise of colistin-resistant MDR ESBL-producing K. pneumoniae, should this antibiotic be routinely introduced to treat infections.
Our study had several limitations. First, the relatively small number of isolates (37 out of 89 possible) is a source of potential bias, as we might have overlooked several phenotypic (antimicrobial susceptibility) and genetic features (phylogenomics, virulence factors and AMR genes) of several potential lineages of MDR ESBL-producing K. pneumoniae in this tertiary care hospital. Secondly, several sequences including those on chromosome and genetic mobile elements can be missing from the draft genomes generated by Illumina short reads. Their use in subsequent genomic analyses can be potentially hampered by this incompleteness [53]. This is evidenced by the failure to assign any replicon to isolates carrying plasmid-mediated AMR genes (i.e., isolates CTMA_1659; CTMA_1664, and CTMA_1667).
In conclusion, our study, despite its limitations, highlights the high genetic variability of MDR ESBL-producing K. pneumoniae in this easternmost province of DRC, calls for additional research across the whole country to determine the prevalence of these MDR ESBL-producing K. pneumoniae isolates, and suggests characterizing AMR genetic determinants, as well as virulence factor genes, that could account for their spread in the country and beyond. In practice, these results call for caution when administering colistin and carbapenems as well as parallel monitoring of the genomic and phenotypic evolution of AMR in DRC. Data Availability Statement: Publicly available datasets were analysed in this study. Raw sequencing data from all K. pneumoniae isolates were submitted to the European Nucleotide Archive (ENA, http://www.ebi.ac.uk/ena) and are available under accession number PRJEB56212 numbers. The data used and/or analysed during the current study are available from the corresponding author upon reasonable request. The DNA sequence of the novel FIA replicon was submitted to the curator of the pMLST database (https://pubmlst.org/plasmid/; submitted on 26 July 2022) for ST assignment.